Microfluidics relates to a multidisciplinary field comprising physics, chemistry, engineering and biotechnology that studies the behavior and flow of fluids at nano and microliter scale inside open or closed microchannels. Microfluidics has enabled the development of so-called ‘lab-on-a-chip’ devices and systems that can process microliter and nanoliter volumes of sample fluid, and perform highly complex and sensitive analytical measurements. ‘Lab-on-a-chip’ indicates the scaling of single or multiple laboratory processes down to a chip-format, which is only millimeters to a few centimeters in size.
Microfluidic-based analysis chips and microfluidic-based diagnostic test chips (hereafter collectively referred to simply as ‘chips’) are used for a variety of biotechnological and diagnostic applications, such as nucleic acid detection, characterization, separation, sizing, and typing; cell manipulation and sorting; biomarker detection; microbial pathogen detection; and miniaturization of chemical synthesis.
Microfluidic-based chips offer many advantages over traditional macro-sized counterparts: Easy handling (less hands-on-time, and less sample and reagents are required) and rapid test results (shorter analysis times). The shorter analysis time and easy handling of microfluidic chips is attractive for diagnostic applications because it enables rapid bedside and doctor's office tests, offering the advantage of rapid test result that can be shared with the patient right away.
However, diagnostic applications require a strict control of the sample fluid flow within the chip in order to ensure the required accuracy and reproducibility of the analysis/test. Typically operations such as mixing; incubation; separation; and detection are performed within the chip, which demands firm control of the sample fluid flow in terms of timing, volumes and flow rates.
Various actuation mechanisms have been developed and are presently used, such as, for example, micropumps, microvalves, and rotary drives applying centrifugal forces. However, the mechanism mostly used in today's products is capillary driven flows, largely due to its simplicity.
In order to take control of the fluid flow in capillary driven microfluidic chips, passive control schemes relying on reproducible geometries and stabile surface properties has been developed. Time gates can be designed to slow down or stop the flow of a predetermined volume for a predetermined time and channels can be designed to have the right capillary force to drive the flow at a predetermined flow rate.
However, for diagnostic applications the variable composition and viscosity of biological samples puts up a major challenge to microfluidic assay development. Typical biological samples include e.g. saliva, urine, serum, plasma and whole blood, and they vary in composition and viscosity from individual to individual as well depending on when the sample was taken. These variations, along with variations in the surface properties of the microfluidic chips, causes variations in timing, volumes and flow rates and can thereby negatively affect the reproducibility of diagnostic microfluidic-based tests.
In order to achieve the firm control needed of the sample fluid flow, in terms of timing, volumes and flow rates in capillary driven microfluidic chips various wettability switches have been developed. A wettability switch allow for manipulation of a solid surface's ability to maintain contact with a liquid. The wettability is determined by a force balance between adhesive and cohesive forces when the solid switch surface is in contact with the liquid. Adhesive forces between the solid and the liquid cause a liquid drop to spread across the switch surface. Cohesive forces within the liquid cause the liquid drop to ball up and avoid contact with the switch surface. The force balance can be manipulated through various influences such as light, temperature, chemistry, electrochemistry or electric field. Within the area of microfluidics some kind of electronic influence is preferred due to its rapid actuation and low interference with the fluid.
One technology used for electronically altering the wettability of a surface is electrochemical electrowetting, where a voltage in the order of one volt is applied between a working electrode and a reference electrode, in electrical contact with each other through an electrolytical fluid, forming an electrochemical double layer at the surface of the work electrode that increases its wettability.
Another technology used for electronically altering the wettability of a surface is electrowetting on dielectric, where tens to hundreds of volts are applied between a working electrode and a reference electrode, electrically isolated from each other, forming a strong electric field that forces the droplet down towards the isolated work electrode surface.
A further technology used for electronically altering the wettability of a surface is electrowetting with ion-doped conducting polymers, where a few volts are applied between a working electrode doped with polar ions, having a hydrophobic and a hydrophilic end, and a reference electrode, making the polar ions shifting orientation and thus shifting the wettability of the work electrode surface.
For the flow control in continuous flow applications electrochemical electrowetting is preferred in its simplicity, amongst others due to a greater wettability change and a better long term stability compared to electrowetting using ion-doped conducting polymers and due to a low voltage need and cost effectiveness compared to electrowetting on dielectric.
Prior art within electrochemical electrowetting has described microfluidic chips having a reference electrode placed in the beginning of a flow path (microfluidic channel) followed by one or a few work electrodes downstream the flow path. The working electrodes are typically hydrophobic thus stopping the fluid in their natural state, but turn hydrophilic and let the fluid pass when a voltage is applied.
However, a major drawback with the electrochemical electrowetting microfluidics technology according to prior art is that the speed of the fluid flow front may rapidly decrease with the distance travelled along the flow path. About one centimeter into the channel, the electrically activated flow may more or less have ceased, thereby impeding activation of a switch.
One example of prior art is described by a research team from Tsukuba in Japan in the article Microfluidic transport based on direct electrowetting, J. Appl. Phys. 96, 835 (2004). The article describes a microfluidic chip of about 20×30 mm with a channel being about 18 mm long. An Au working electrode is covering the bottom of the channel and an Ag/AgCl reference electrode is placed upstream the working electrode.
In the article the research team conclude that when applying a voltage between the working electrode and the reference electrode the fluid front starts to move along the working electrode. However, they conclude that the speed of the fluid front decreases with the distance travelled in the channel and that the movement finally cease. In fact the research team concludes that the fluid front never reaches through the about 18 mm long channel.
The research team conclude that the decrease of the flow rate and final cease of the flow are due to the viscous resistance, which is said to increase linearly with the distance travelled. The research team further conclude that by working with microfluidic chips of about 10×10 mm this limitation might be acceptable.
However, a channel length limitation of about 1 cm is for practical reasons limiting the amount of sample fluid that is possible to handle (in terms of timing, volumes and flow rates), to nanoliter volumes rather than microliter volumes. When targeting applications within medical diagnostics this is a major drawback, since most point of care tests require a sample volume in the microliter range in order to produce accurate and reproducible results.
In reality, the channel length limitation is actually shorter than 1 cm when using the working electrode(s) for driving a flow within the channel. When targeting applications within medical diagnostics flow velocities of tens of millimeter per minute are typically required. Due to the rapid decrease of the flow velocity, the velocity of the fluid front is below what is required already a few millimeters into the channel.
The channel length limitation is for practical reasons also limiting the number of working electrodes that are possible to place along the flow path, given a certain minimal working electrode size possible to produce cost effectively (for instance the smallest working electrode possible to produce with a metal sputter mask instead of photolithography). When targeting medical diagnostics, multiple operations according to above need to be performed with firm and fine-tuned flow control. However, a limited number of fairly large working electrodes, relative to the length of the channel, is thus limiting the number of operations and the possibility of fine-tuned flow control.
Altogether, due to a channel length limitation of about 1 cm or even less, prior art has a limited ability to handle microliter volumes of sample fluid with the firm control of timing, volumes and flow rates required for many applications within medical diagnostics.